PROJECT SUMMARY: Persistent pain costs about $650 billion annually, making it a top NIH priority. All current local anesthetics are short acting and disabling due to complete sensory and motor blockade. In the absence of suitable alternatives, an epidemic of opioid overuse, abuse, and life-threatening complications has occurred. LONG- TERM GOALS: Develop a safe and effective carbonic anhydrase-8 (CA8)-mediated Long Acting Local Anesthetic (LALA) when administered to peripheral somatosensory nerves produces profound analgesia without motor blockade, loss of desirable sensory function or systemic toxicity. HYPOTHESIS: CA8 biotherapy delivered to subsets of primary afferent neurons (e.g., Nav1.8 or Advillin expressing) can produce intense long- lasting local analgesia without loss of protective sensory functions, motor blockade, or toxicity. RATIONALE: We show in genetic studies CA8 regulates analgesia and analgesic responses. Moreover, we show that sciatic nerve injection with AAV-CA8 mediated overexpression in mice provides profound analgesia (equivalent >200mg of oral morphine in 60kg adult), without motor blockade or gross clinical pathology. Herein, we will develop and test the first novel proprietary CA8-mediated LALA that selectively targets Nav1.8 or Advillin expressing primary afferents. HSV was used in prior clinical trials without toxicity and is currently the only FDA approved gene therapy. This Staged Innovation Proposal (PAR-15-071) includes R21 Phase: AIM 1 (Yr. 1) we will develop and produce highly purified high titer novel HSV CA8-based (vHCA8) biotherapeutics, QC and test for preclinical validation of structure and function in vitro including potency using inhibition of pITPR1, Ca++ release, neuronal excitability and cellular toxicity. R33 Phase: AIM 2 (Yrs. 2 & 3) will assess analgesic dose- response related to tissue specificity of these biotherapeutics in vivo by examining initial efficacy (analgesia) testing using mechanical (von Frey), thermal (Hargreaves); and non-reflexive sensory and motor testing. We will also assess novel routes familiar to practitioners including peripheral nerve `block' compared to intradermal HSV administration. Selective (Nav1.8) and non-selective (Advillin) vHCA8-mediated CA8 overexpression can assess pharmacodynamics using pITPR1, Ca++ release, and neuronal excitability in vitro. Additionally, we will run initial CA8 biotherapeutic efficacy and safety studies using the well-accepted model of neuropathic pain (spinal nerve ligation). Clinical safety assessments will include toxicity related to mechanism (gross inspection, organ weights; histopathology; CA8-specific sciatic nerve, DRG and spinal cord toxicity; neuronal, glial and microglial apoptosis); and off-target clinical measures and histopathology. We expect targeted CA8 somatosensory overexpression will provide a proof-of-concept regarding the role of primary afferents in chronic pain, and define initial therapeutic indications of CA8 biotherapeutics. At the conclusion of these preclinical studies we expect patent filings facilitating commercialization, IND-enabling GLP safety studies; followed by clinical trials. CA8 biotherapeutics represent a potentially transformational approach to address major unmet needs in chronic pain, improving function, quality-of-life and minimizing opioid usage by millions of patients.